Mutagenic Potential of Stereoisomeric Bay Region (+)- and (-)-cis-anti-Benzo[a]pyrene Diol Epoxide-N 2 -2′-deoxyguanosine Adducts in Escherichia coli and Simian Kidney Cells ² Andrea Fernandes, ‡ Tongming Liu, § Shantu Amin, | Nicholas E. Geacintov, § Arthur P. Grollman, ‡ and Masaaki Moriya* ,‡ Department of Pharmacological Sciences, State UniVersity of New York, Stony Brook, New York 11794-8651, Department of Chemistry, New York UniVersity, New York, New York 10003, and American Health Foundation, Valhalla, New York 10595 ReceiVed February 19, 1998; ReVised Manuscript ReceiVed May 20, 1998 ABSTRACT: We have investigated the mutagenic potential of site-specifically positioned DNA adducts with (+)- and (-)-cis-anti stereochemistry derived from the binding of r7,t8-dihydroxy-t9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) to N 2 -2′-deoxyguanosine (G 1 or G 2 ) in the sequence context 5′ TCCTCCTG 1 G 2 CCTCTC. BPDE-modified oligodeoxynucleotides were ligated to a single-stranded DNA vector and replicated in Escherichia coli or simian kidney (COS7) cells. The presence of (+)- or (-)-cis adduct strongly reduced the yield of transformants in E. coli, and the yield was improved by the induction of SOS functions. Both adducts were mutagenic in E. coli and COS cells, generating primarily G f T transversions. In E. coli, the (-)-cis adduct was more mutagenic than the (+)-cis adduct, while in COS cells, both adducts were equally mutagenic. These results were compared with those obtained with stereoisomeric (+)- and (-)-trans adducts [Moriya, M., et al. (1996) Biochemistry 35, 16646- 16651). In E. coli, cis adducts, especially (-)-cis adducts, are consistently more mutagenic than the comparable trans adduct. In COS cells, trans adducts yield higher frequencies of mutations than the two cis adducts and, with the exception of the high-mutation frequency associated with the (+)-trans adduct at G 2 , relatively small differences in mutation frequencies are observed for the three other adducts. In E. coli, mutation frequency is a pronounced function of adduct stereochemistry and adduct position. These findings suggest that the fidelity of translesional synthesis across BPDE-dG adducts is strongly influenced by adduct stereochemistry, nucleotide sequence context, and the DNA replication complex. Polycyclic aromatic hydrocarbons (PAHs) 1 are ubiquitous environmental pollutants that can be activated by cellular multifunction oxidases to produce a variety of oxygenated metabolites (1). The diol epoxide derivatives bind covalently to cellular DNA, thus inducing mutations and initiating tumorigenesis in animal model systems (2). Benzo[a]pyrene (BP), one of the most widely studied PAH compounds (3), is metabolized to oxygenated derivatives, including the biologically important (+)-enantiomer of r7,t8-dihydroxy- t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE, or BPDE). The (+)-7R,8S,9S,10R-enantiomer [(+)-anti- BPDE] is more tumorigenic than the (-)-7S,8R,9R,10S- enantiomer [(-)-anti-BPDE] when applied to mouse skin and in the newborn mouse lung model (4, 5). In mammalian cell systems, (+)-anti-BPDE is more mutagenic than the (-)- enantiomer (6-9), while in bacteria the relative mutagenici- ties of the two enantiomers depend on the test system utilized (6, 8). The tumorigenic activities of BPDE correlate with the formation of covalent adducts with cellular DNA (10, 11). Both BPDE enantiomers react with native DNA by binding predominantly to the exocyclic amino group of 2′-deoxygua- nosine residues (dG) by trans or cis addition of N 2 -dG to the C10-position of anti-BPDE (12-15). While trans addition is dominant, smaller quantities of cis-N 2 -dG as well as N 6 -2′-deoxyadenosine adducts also are formed (15). When cells are treated with BPDE, a variety of DNA adducts is produced. Due to the stereochemical diversity of these adducts, it is not feasible to relate the observed mutagenic spectra and mutagenic specificities to a particular type of lesion. Nevertheless, useful observations have been made in random modification experiments using racemic mixtures of anti-BPDE (16-21), the optically resolved (+)- anti-BPDE (22, 23), or (-)-anti-BPDE enantiomers (9). The most frequently observed point mutations recorded in these studies were G f T transversions: G f A and G f C base substitutions also were observed, but generally were less abundant. ² This research was supported by NIH Grants ES04068 and CA17395 (to A.P.G.) and CA 20851 (to N.E.G.). * To whom correspondence should be directed. ‡ State University of New York, Stony Brook. § New York University. | American Health Foundation. 1 Abbreviations: BP, benzo[a]pyrene; BPDE, r7,t8-dihydroxy- t9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; CD, circular dichroism; PAH, polycyclic aromatic hydrocarbons; ss, single stranded; TEAA, triethylammonium acetate. 10164 Biochemistry 1998, 37, 10164-10172 S0006-2960(98)00401-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/25/1998